The vitamin K oxidoreductase (VKOR) plays a critical role in hemostasis because it provides the cofactor required for the carboxylation of vitamin K-dependent (VKD) hemostatic proteins. Vitamin K from the diet circulates in blood, where it is taken up by tissues and delivered to the endoplasmic reticulum to be used in carboxylation. During carboxylation, reduced vitamin K is used to drive the conversion of clusters of Glus to carboxylated Glus in VKD proteins, rendering them functional for hemostasis. Carboxylation results in vitamin K oxidation, and VKOR reduces the oxidized vitamin K for continuous carboxylation by a mechanism that is largely unknown. VKOR, which is the target of anticoagulant drugs like warfarin, becomes inactivated during vitamin K reduction and therefore also requires continuous recycling, which is accomplished by a redox protein not yet identified. Thus, VKOR participates with multiple components in the process of carboxylation;however, how it interacts with these proteins to accomplish efficient carboxylation is unknown, as is the identity of some of the proteins. We have developed mammalian cells expressing recombinant carboxylation components as a model system to understand this process, and this system provides a unique set of tools for defining VKOR function. Our Specific Aims are to: 1) Determine the importance of a thioredoxin reductase isoform to VKOR activity and VKD protein carboxylation. We found that vitamin K availability limits carboxylation in cells and that VKOR overexpression only caused a small increase in carboxylation, which we hypothesize is due to saturation of the redox protein required for VKOR activity. We identified a thioredoxin reductase isoform as a VKOR-interacting protein that is required for carboxylation, and will test our hypothesis by determining how changes in the level of this isoform alter carboxylation in cells. 2) Define how mutations in cytoplasmic VKOR sequences confer resistance to warfarin. Unexpectedly, VKOR mutations known to cause warfarin resistance in humans show sensitivity to this drug when analyzed in vitro. We hypothesize that the difference is due to the impact of VKOR-interacting proteins like the thioredoxin reductase isoform or a fatty acid binding protein that we also identified and showed is important to carboxylation. We will test our hypothesis by determining the effect of these proteins on VKOR susceptibility to warfarin. 3) Determine how VKOR supplies reduced vitamin K to the carboxylase. Vitamin K recycling between VKOR and the carboxylase is very efficient, raising the question of whether recycling is facilitated by complex formation between the two enzymes. We will test for the existence of a complex using in vitro VKD protein carboxylation assays we developed as well as FRET in cells. These studies will provide fundamental contributions to our understanding of VKOR physiology in VKD protein carboxylation that will lead to the development of superior anticoagulants and improved production of VKD proteins for therapeutic use.